Visualization of Arabidopsis root system architecture in 3D by refraction-contrast X-ray micro-computed tomography

Microscopy, 2021, 536–544 doi:https://doi.org/10.1093/jmicro/dfab027 Advance Access Publication Date: 30 July 2021 Article Visualization of Arabidopsis root system architecture in 3D by refraction-contrast X-ray micro-computed tomography Tomofumi Kurogane1 , Daisuke Tamaoki2 , Sachiko Yano3 , Fumiaki Tanigaki3 , Toru Shimazu4 , Haruo Kasahara5 , Daisuke Yamauchi6 , Kentaro Uesugi7 , Masato Hoshino7 , Seiichiro Kamisaka2 , Yoshinobu Mineyuki6 and Ichirou Karahara2,* 1 Graduate School of Science and Engineering for Education, University of Toyama, 3190 Gofuku, Toyama 930-8555, Japan Faculty of Science, University of Toyama, 3190 Gofuku, Toyama 930-8555, Japan 3 Human Spaceflight Technology Directorate, Japan Aerospace Exploration Agency, 2-1-1 Sengen, Tsukuba 305-8505, Japan 4 Space Utilization Promotion Department, Japan Space Forum, 3-2-1 Kandasurugadai, Tokyo 101-0062, Japan 5 ISS Utilization and Operations Department, Japan Manned Space Systems Corporation, 1-1-26 Kawaguchi, Tsuchiura 300-0033, Japan 6 Department of Life Science, Graduate School of Science, University of Hyogo, 2167 Shosha, Himeji, Hyogo 671-2280, Japan 7 Scattering and Imaging Division, Japan Synchrotron Radiation Research Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5198, Japan 2 *To whom correspondence should be addressed. E-mail: Received 26 April 2021; Revised 12 July 2021; Editorial Decision 12 July 2021; Accepted 15 July 2021 Abstract Plant roots change their morphological traits in order to adapt themselves to different environmental conditions, resulting in the alteration of the root system architecture. To understand this mechanism, it is essential to visualize the morphology of the entire root system. To reveal effects of long-term alteration of gravity environment on root system development, we have performed an experiment in the International Space Station using Arabidopsis plants and obtained dried root systems grown in rockwool slabs. The X-ray computed tomography (CT) technique using industrial X-ray scanners has been introduced to visualize the root system architecture of crop species grown in soil in 3D non-invasively. In the case of the present study, however, the root system of Arabidopsis is composed of finer roots compared with typical crop plants and rockwool is also composed of fibers having similar dimension to that of the roots. A higher spatial resolution imaging method is required for distinguishing roots from rockwool. Therefore, in the present study, we tested refraction-contrast X-ray micro-CT using coherent X-ray optics available at the beamline of the synchrotron radiation facility SPring-8 for bio-imaging. We have found that a wide field of view but with low resolution obtained at the experimental Hutch 3 of this beamline provided an overview map of the root systems, while a narrow field of view but with high resolution obtained at the experimental Hutch 1 provided an extended architecture of the secondary roots, by a clear distinction between roots and individual rockwool fibers, resulting in the successful tracing of these roots from their basal regions. Key words: Arabidopsis, root system architecture, SPring-8, 3D observation, X-ray micro-CT, synchrotron radiation © The Author(s) 2021. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: 536 T. Kurogane et al. X-ray micro-CT of Arabidopsis root system Introduction Methods and materials Plant materials and growth conditions The present experiment was performed during the preparation of the Space Seed experiment. Growth conditions are basically the same as described previously [14], while the plant materials and the employed instrument were as follows. Twenty-four seeds of Arabidopsis (A. thaliana (L.) Heynh.) Landsberg erecta (Ler) were sterilized and sown on a rockwool slab (W × D × H = 50 × 42 × 10 mm) (Nichias Corp, Tokyo, Japan) using gum Arabic and germinated in a polycarbonate growth chamber having the outer dimensions of W × D × H = 60 × 50 × 60 mm, and the dimensions of its inner void space were W × D × H = 56 × 46 × 48 mm (Fig. 1). The rockwool slab was covered with a transparent plastic plate and growth chambers were installed in the prototype of the plant experimental units, which was designed for experiments in the International Space Station [14]. Plants were illuminated laterally with light-emitting diode matrix [16], and light intensity was 29 µmol m−2 s−1 when measured at the bottom center of the growth chamber. Plants used for the following experiment were grown for 46 days. After that, the plants and rockwool slabs were dried at room temperature with silica gel in a desiccator until observation. The reason why dried root systems were used in the present study is that the plants which terminated their life cycle in the Space Seed experiment were naturally dried in the growth chambers in space. After sample return from space, shoots of the plants were removed before the observation. Typical plants during its development are shown in Fig. 1. Refraction contrast X-ray micro-CT Refraction contrast X-ray micro-CT was performed at the experimental hutches (Hutch 3 and 1), where different spatial resolutions are available, of the beamline BL20B2 of the SPring-8 synchrotron radiation facility at Japan Synchrotron Radiation Research Institute, according basically to the method described by Karahara et al. [17]. Its experimental setup is shown in Fig. 2, and a brief of the method is as follows. The Hutch 3 and 1 are located 42 and 206 m, respectively, from the bending magnet X-ray source. The X-ray energy was adjusted to 25 keV. The images consecutively projected on the fluorescent screen were recorded by a CMOS camera (ORCA-Flash 4.0; Hamamatsu Photonics KK, Hamamatsu, Japan) (Fig. 2a). The image sizes obtained at the Hutch 1 and Hutch 3 were 2048 × 2048 pixels (approximately 5 × 5 mm) and 2048 × 556 pixels, (approximately 50 × 15 mm), respectively. A series of 900 and 3000 projections were recorded over 180◦ for Hutch 1 and 3 observation, respectively. Because thickness of the rockwool slab Fig. 1. Pictures of typical Arabidopsis plants grown on a rockwool slab in a polycarbonate growth chamber. (a) Plants on Day 12. Rosette leaves are growing. The surface of the rockwool slab is seen underneath the plants. (b) Plants on Day 32. Flowers are forming. Internal length of the front side of the chamber is 46 mm. Red arrowheads indicate the rockwool slabs. The plant root system provides a basis for supporting and anchoring the shoot system for growth by uptaking water and nutrients from the soil. The plant root system adapts itself to the surrounding soil environment changing its architecture [1]. This capability of the plant root system is called root system plasticity [2]. Understanding the mechanism of root plasticity is important for the optimization of plant cultivation conditions under a given environmen (...truncated)